Novel Role of Cold/Menthol-sensitive Transient Receptor Potential Melastatine Family Member 8 (TRPM8) in the Activation of Store-operated Channels in LNCaP Human Prostate Cancer Epithelial Cells

Abstract

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I cold/menthol ) that shows inward rectification and high Ca 2؉ selectivity, which are dramatically different properties from "classical" TRPM8-mediated I cold/menthol . Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I cold/menthol and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca 2؉ release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca 2؉ release channel, potentially involved in a number of Ca 2؉ -and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis. Mammalian homologues of the Drosophila transient receptor potential (TRP)7 channel, which initially emerged as a channel specifically linked to phospholipase C-catalyzed inositol phospholipid breakdown signaling pathways, have now grown into a broad family of channelforming proteins displaying extraordinarily diverse activation mechanisms (for reviews, see Refs. 1-5). At present, these channels are grouped into six subfamilies based on structural homology and have been given a standard nomenclature (5).A number of mammalian TRPs show a unique mode of gating, in response to thermal stimuli as well as to the chemical imitators of burning and cooling sensations, capsaicin and menthol, respectively. As such, they represent a group of thermal receptors covering a wide range of physiological temperatures. Most thermal receptors belong to the vanilloid TRP subfamily (TRPV, Ref. 6) including warm-sensitive (Ͻ40°C) TRPV3 (7-9) and heat-and capsaicin-sensitive TRPV1 (Ͼ43°C) (10) and TRPV2 (Ͼ52°C) (11). In contrast, sensitivity to cooling temperatures (Ͻ22°C) and menthol is mediated by a structurally distant thermal receptor, TRPM8, belonging to the melastatine (TRPM) subfamily of TRP channels (12, 13); the ankyrin transmembrane protein 1 (ANKTM1 or TRPA1) is involved in the detection of noxious cold (14).Consistent with their role in the sensation of distinct physiological temperatures, thermal receptors are mostly expressed in subsets of...

TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

“…Later studies have reported similar menthol-induced Ca 2ϩ release in TRPM8-expressing LNCaP cells and dorsal root ganglion neurons (4,7,8), leading to the hypothesis that TRPM8 can function as an intracellular Ca 2ϩ -release channel. Further support to this idea came from immunocytochemical stainings indicating that TRPM8 is significantly or even exclusively located on intracellular membranes in LNCaP cells (7,8). However, our present data indicate that these results should be interpreted with caution.…”

“…3F). As previous studies reporting that endogenous TRPM8 in LNCaP cells is to a large extent or even fully restricted to intracellular compartments used the commercial anti-TRPM8 antibodies (7,8), we repeated immunocytochemical staining of LNCaP using our new anti-TRPM8 antibody. In contrast to the results from these previous studies, the staining pattern that we obtained was consistent with a principally plasma- lemmal localization of TRPM8 (Fig.…”

“…Yet, the function of TRPM8 in non-sensory cells and particularly its relation to the pathophysiology of cancer cells are currently unknown. Recent studies have suggested that TRPM8, besides its established function as a plasmalemmal channel, can also be found in the ER membrane of the androgenresponsive LNCaP prostate cell line, where it may function as a menthol-and cold-sensitive intracellular Ca 2ϩ -release channel (7,8). In addition, menthol-induced release of Ca 2ϩ from intracellular stores has been reported in other different cell types, including skeletal muscle (9), tracheal epithelial cells (10), and dorsal horn neurons (11).…”

PSA reduces prostate cancer cell motility by stimulating TRPM8 activity and plasma membrane expression

“…In addition, icilin did not trigger any [Ca 2 þ ] i Figure 1 PSA activates TRPM8. (a) Representative time courses of cold-activated (22 1C) I TRPM8 under control condition (white dots) or after overnight pre-treatment with 40 ng/ml PSA (black dots) in the HEK-TRPM8-inducible cell line, cultured as previously described (Thebault et al, 2005). TRPM8 expression was induced with tetracycline 24 h before the experiments.…”

“…(b) Cumulative data of the [Ca 2 þ ] i in HEK-TRPM8 cells in control (black column) or after overnight pre-treatment with 40 ng/ml PSA (gray column) after icilin-evoked TRMP8 Ca 2 þ entry. [Ca2 þ ]i was measured using fura-2, as previously described (Thebault et al, 2005;Bidaux et al, 2007). The same extracellular solution was used as in electrophysiological experiments.…”